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Proapoptotic Function of the Retinoblastoma Tumor
Suppressor Protein
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Ianari, Alessandra, Tiziana Natale, Eliezer Calo, Elisabetta
Ferretti, Edoardo Alesse, Isabella Screpanti, Kevin Haigis,
Alberto Gulino, and Jacqueline A. Lees. “Proapoptotic Function
of the Retinoblastoma Tumor Suppressor Protein.” Cancer Cell
15, no. 3 (March 2009): 184-194. Copyright © 2009 Elsevier Inc.
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Cancer Cell
Proapoptotic Function
of the Retinoblastoma Tumor Suppressor Protein
Alessandra Ianari,1,2 Tiziana Natale,2 Eliezer Calo,1 Elisabetta Ferretti,2 Edoardo Alesse,3 Isabella Screpanti,2
Kevin Haigis,4 Alberto Gulino,2,5,* and Jacqueline A. Lees1,*
H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA
of Experimental Medicine, La Sapienza University of Rome, 00161 Rome, Italy
3Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy
4Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA
5Neuromed Institute, 86077 Pozzilli, Italy
*Correspondence: (A.G.), (J.A.L.)
DOI 10.1016/j.ccr.2009.01.026
The retinoblastoma protein (pRB) tumor suppressor blocks cell proliferation by repressing the E2F transcription factors. This inhibition is relieved through mitogen-induced phosphorylation of pRB, triggering E2F
release and activation of cell-cycle genes. E2F1 can also activate proapoptotic genes in response to genotoxic or oncogenic stress. However, pRB’s role in this context has not been established. Here we show that
DNA damage and E1A-induced oncogenic stress promote formation of a pRB-E2F1 complex even in proliferating cells. Moreover, pRB is bound to proapoptotic promoters that are transcriptionally active, and pRB is
required for maximal apoptotic response in vitro and in vivo. Together, these data reveal a direct role for pRB
in the induction of apoptosis in response to genotoxic or oncogenic stress.
The retinoblastoma gene (RB1), a member of the pocket protein
family with p107 and p130, was the first known tumor
suppressor. The retinoblastoma protein (pRB) is targeted by
the transforming proteins of the DNA tumor viruses (e.g., adenoviral E1A), and it is functionally inactivated in a large proportion
of human tumor cells due to mutations of either the RB1 gene
itself or its upstream regulators (Trimarchi and Lees, 2002).
pRB’s tumor-suppressive activity is thought to be largely dependent upon its ability to directly bind members of the E2F family of
transcription factors and prevent them from promoting transcription of genes required for cell proliferation (Trimarchi and Lees,
2002). This inhibition can occur via two distinct mechanisms:
pRB binds to sequences within E2F’s transactivation domain
and inhibits its function, and the resulting pRB-E2F complex
recruits a number of transcriptional corepressors, including
histone deacetylases (HDACs), methyltransferases, and polycomb group proteins to actively repress the promoters of E2F
target genes.
In normal cells, pRB’s repressive activity is controlled by its
cell-cycle-dependent phosphorylation (Trimarchi and Lees,
2002). In response to mitogenic signaling, pRB is sequentially
phosphorylated by the Cdk complexes cyclin D-Cdk4/6 and
cyclin E-Cdk2. This phosphorylation is sufficient to induce pRB
to release E2F, thereby allowing activation of E2F-responsive
genes in late G1. However, phosphorylated pRB (ppRB) persists
in the nucleus through the remainder of the cell cycle until it is
dephosphorylated by protein phosphatase 1 at the end of
mitosis (Ludlow et al., 1993). It is widely assumed that ppRB is
functionally inactive and that dephosphorylation restores pRB
to the active state. The majority of human tumors carry mutations
that disable pRB-mediated repression of E2F (Sherr and McCormick, 2002). These mutations either inactivate the RB1 gene
itself or promote pRB phosphorylation in the absence of normal
mitogenic signals through activation of the cyclin D-Cdk4/6
kinases or inactivation of the Cdk inhibitor p16. These changes
result in the inappropriate release of E2F, thereby inducing transcriptional activation of E2F target genes and consequently cell
Retinoblastoma protein (pRB) function is disrupted in many human tumors through either inactivation of the RB1 gene or
alterations in its upstream regulators. pRB’s tumor-suppressive activity is at least partially dependent upon its ability to
arrest cells through E2F inhibition. Our data here now establish a second role for pRB as a stress-induced activator of
apoptosis. Notably, pRB’s ability to promote either arrest or apoptosis seems to be context dependent, with apoptosis
being favored in proliferating cells. This finding has the potential to explain why cells are typically more resistant to
apoptosis when in the arrested state. Most importantly, our observations suggest that RB1 status will influence tumor
response to chemotherapy by impairing both the arrest and apoptotic checkpoint responses.
184 Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc.
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
It is well established that E2F1, among other E2F family
members, also contributes to the induction of apoptosis in
response to either DNA damage or oncogenic stress (Iaquinta
and Lees, 2007). This is thought to be a critical event in suppressing the formation of tumors. Work from many laboratories has
shown that genotoxic stress induces E2F1 recruitment to the
promoters of proapoptotic genes including p73 and Caspase
7, coincident with their transcriptional activation, even in cells
that retain wild-type pRB (Pediconi et al., 2003). This led us to
consider how pRB influences DNA damage-induced apoptosis.
The prevailing view is that pRB is an antiapoptotic regulator.
Early support for this model came from the finding that several
tissues in Rb mutant mice display both ectopic proliferation
and apoptosis (Jacks et al., 1992). However, it is now clear
that much of this apoptosis is non-cell autonomous, resulting
from a proliferation defect in the extraembryonic tissues (de
Bruin et al., 2003; Wenzel et al., 2007; Wu et al., 2003). Analysis
of tissue-specific Rb mutant models reinforces the notion that
pRB plays a much more nuanced role in apoptosis. Loss of
pRB in neuronal tissue (MacPherson et al., 2003), lung (MasonRichie et al., 2008; Wikenheiser-Brokamp, 2004), skin (Ruiz
et al., 2004), and intestine (Haigis et al., 2006; Wang et al.,
2007) drives ectopic proliferation but has no effect on apoptosis.
In contrast, Rb inactivation in the lens (de Bruin et al., 2003) and
myoblasts (Huh et al., 2004) does induce apoptosis, but this is
specifically observed in the differentiating cells. Thus, taken
together, these mouse studies support two general conclusions:
first, in many different settings, Rb inactivation can induce inappropriate proliferation without triggering apoptosis, and second,
when apoptosis is observed, it seems to result from an inability to
cease proliferation and undergo terminal differentiation.
pRB’s apoptotic role has also been analyzed in established
tissue culture cell lines. However, these studies have yielded
conflicting results: some conclude that pRB suppresses
apoptosis (Almasan et al., 1995; Bosco et al., 2004; Knudsen
et al., 2000), whereas others suggest that it is proapoptotic (Araki
et al., 2008; Bowen et al., 1998, 2002; Knudsen et al., 1999).
None of these studies addresses the molecular basis for the
observed role of pRB. In this study, we investigated how pRB
influences the ability of E2F to induce apoptosis in response to
genotoxic stress.
Stabilization of the pRB-E2F1 Complex in Response
to DNA Damage
In cells committed to die by apoptosis in response to either DNA
damage or oncogenic stress, E2F1 transcriptional activity is
directed toward promoters of a subset of apoptotic genes that
include Caspase 7, p73, and Apaf1 (Iaquinta and Lees, 2007).
Thus, we hypothesized that DNA damage must somehow
inactivate pRB’s repressive function to allow the release of transcriptionally active E2F1. To test this hypothesis, we assessed
the binding of pRB to E2F1 both before and after doxorubicin
treatment of human T98G cells. However, contrary to our expectations, we found that the pRB-E2F1 complex was stabilized
by this genotoxic stress (Figure 1A). Notably, T98G cells are
p53 defective and therefore treatment with doxorubicin causes
them to accumulate in G2/M (Figure 1B). Thus, we can conclude
that the observed DNA damage-induced formation of the pRBE2F1 complex is neither dependent on p53 nor simply an indirect
consequence of a G1 arrest.
pRB’s ability to bind to E2F is normally limited to the early
stages of the cell cycle when pRB exists in the hypophosphorylated form. Therefore, it was surprising to observe pRB-E2F1
complexes in a population highly enriched for G2/M phase cells.
To more directly address the influence of cell-cycle phasing, we
used serum deprivation and readdition to generate two populations of T98G cells that were greatly enriched for either G0/G1
(70%) or proliferating (95% S or G2/M phase) cells (Figure 1C)
and then treated these with doxorubicin. Consistent with the
known cell-cycle-dependent phosphorylation of pRB, the pRB
protein was present in its slower mobility form in untreated
proliferating cells, and it bound little E2F1 (Figure 1C). Notably,
doxorubicin treatment was still able to induce formation of the
pRB-E2F1 complex in this proliferating population (Figure 1C).
This occurred independently of any change in total levels of
E2F1 protein (Figure 1C), and it correlated with full activation of
the apoptotic program as judged by PARP-p85 induction (data
not shown). Importantly, the G0/G1 cells within the proliferating
population cannot fully account for this DNA damage-induced
pRB-E2F1 complex because the treated proliferating cells and
untreated G0/G1 cells had comparable levels of pRB-associated
E2F1 (Figure 1C), but the fraction of G0/G1 cells in the enriched
populations differed by 14-fold (5% versus 70%). Thus, these
data show that pRB-E2F1 complexes can form in S/G2/M phase
cells in response to DNA damage.
pRB is sequentially phosphorylated by the cyclin D-Cdk4/6
and cyclin E-Cdk2 complexes, and this is thought to disrupt
the interaction between pRB and E2F. Since DNA damage
causes pRB to bind to E2F1 irrespective of cell-cycle phase,
we wished to determine whether ppRB could participate in this
complex. To this end, we generated enriched populations of
G0/G1 and proliferating T98G cells, exposed them to either
ionizing radiation (IR) or doxorubicin (see schema in Figure 1D,
left panel), and then assessed both the levels and E2F1-binding
properties of ppRB using antibodies that specifically recognize
known Cdk phosphorylation sites within pRB, pSer780,
pSer795, and pSer807–811. Fluorescence-activated cell sorting
(FACS) analysis confirmed the high degree of enrichment of the
G0/G1 and proliferating populations both before and after IR or
doxorubicin treatment (Figure 1D, right panel). As expected,
ppRB was present at much higher levels in the proliferating
versus the G0/G1 cells as judged by both the immunoprecipitation (IP) of ppRB and the mobility shift of the total pRB
(Figure 1E). We found that a small subset of the E2F1 coimmunoprecipitated with ppRB before treatment, and IR and doxorubicin both increased this level (Figure 1E, left panel). This
increased binding occurred independently of any change in the
total levels of E2F1 (Figure 1E, right panel). Interestingly, IR
and doxorubicin increased the levels of ppRB in the G0/G1 population (Figure 1E, compare lane 1 with lanes 2 and 3), even
though neither treatment altered the cell-cycle distribution of
these cells (Figure 1D, right panel).
The increased level of ppRB in the treated G0/G1 cells clearly
contributes to, but seems insufficient to fully account for, the
increased level of E2F1 in the ppRB immunoprecipitates.
Indeed, for all of the cell-cycle fractions, we clearly recovered
Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc. 185
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
tail disrupts the ppRB-E2F1 complex, leading to an underestimation of its levels, or whether hypophosphorylated or other
phosphorylated pRB species are the main constituent of this
complex. To distinguish between these possibilities, we conducted a reciprocal IP. Specifically, we immunoprecipitated
E2F1 from T98G cells before or after doxorubicin treatment
and then screened for associated pRB by western blotting
with an antibody that recognizes all forms of pRB (Figure 1F).
In the untreated cells, the E2F1 immunoprecipitate contained
a single pRB species. In contrast, doxorubicin caused E2F1 to
bind two distinct pRB bands that were present at approximately
equal levels. One of these comigrated with the single pRB
species seen in the untreated cells, while the other had a slower
mobility characteristic of ppRB. To verify this, we stripped and
reprobed the blot with the anti-ppRB antibody cocktail. This
recognized only the slower migrating pRB species specific to
the doxorubicin-treated cells, confirming that this was ppRB.
Based on the relative levels of the two bands in the anti-pRB
blot, we conclude that ppRB accounts for at least half of the
E2F1-associated pRB activity in the doxorubicin-treated T98G
cells. Taken together, these experiments show that DNA
damage induces formation of pRB-E2F1 complexes in both arrested and proliferating cells and that ppRB is able to participate
in this complex.
Figure 1. DNA Damage Promotes Formation of a pRB-E2F1
Complex in Proliferating Cells
(A and B) Asynchronous T98G cells untreated ( ) or treated with 2 mM doxorubicin (doxo) for 24 hr (+) were screened by immunoprecipitation (IP) using
an antibody against pRB followed by western blotting (WB) to assess levels
of pRB and associated E2F1 and P/CAF (A) or assayed for cell-cycle distribution by FACS analysis (B). Bars in (B) represent the mean of three independent
experiments ± SD.
(C) T98G cells were highly enriched for G0/G1 or S/G2/M (prolif.) cells as determined by FACS (right panel) by culturing in 0.1% FBS for 72 hr and then maintaining in 0.1% FBS or replating in 20% FBS for 16 hr. The cells were collected
(samples 1 and 3) or treated with 2 mM doxo for additional 48 hr (samples 2 and
4) and then assayed in parallel for pRB-E2F1 complexes by IP-WB (left panel).
(D and E) Enriched populations of G0/G1 or proliferating T98G cells were
untreated (samples 1 and 4), irradiated (IR, 10 Gy) for 1 hr (samples 2 and 5),
or treated with 2 mM doxo for 24 hr (samples 3 and 6) as indicated (D, left
panel). These samples were analyzed for cell-cycle phasing by FACS (D, right
panel), the presence of ppRB-E2F1 complexes by IP with either IgG control or
ppRB antibodies and then WB for pRB or associated E2F1 (E, left panel), or the
levels of total pRB and E2F1 of the input lysates by WB (E, right panel).
(F) Asynchronous T98G cells, untreated ( ) or treated with 2 mM doxo for
24 hr (+), were screened for the presence of ppRB-E2F1 complexes by IP
with either IgG control or E2F1 antibodies and then WB, first for pRB and
subsequently (after stripping the blot) for ppRB. A bubble in the blot yielded
the nonspecific signal (*).
a smaller fraction of total E2F1 in ppRB immunoprecipitates
(Figure 1E) versus total pRB immunoprecipitates (Figure 1C). It
was unclear whether the phosphospecific pRB antibody cock186 Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc.
pRB Participates in Transcriptional Activation
of Proapoptotic Genes in Response to Stress
Histone acetyltransferases (HATs) and HDACs are known to play
key roles in mediating the transcriptional properties of pRB and
the E2F proteins (Frolov and Dyson, 2004). The pRB-E2F
complex is thought to act as a repressor of classic E2F target
genes through recruitment of HDACs, while HATs acetylate
E2F1 and promote its transcriptional activity. Importantly, DNA
damage triggers the HAT P/CAF to bind and acetylate E2F1
(Ianari et al., 2004), and this modification is required for E2F1
association with proapoptotic promoters (Pediconi et al.,
2003). Given these observations, we screened for the presence
of P/CAF in pRB immunoprecipitates in untreated versus doxorubicin-treated cells (Figure 1A). We found that DNA damage
promotes P/CAF-pRB complex formation (Figure 1A). This
raised the possibility that pRB might participate in a transcriptionally active complex under proapoptotic conditions.
To understand the transcriptional relevance of the DNA
damage-induced pRB-E2F1 complex, we examined the regulation of representative cell-cycle control and proapoptotic E2F1responsive genes in DNA-damaged T98G cells. This analysis
revealed that doxorubicin caused a differential response of these
two target gene classes: activation of the proapoptotic genes
Caspase 7 and p73 and repression of the cell-cycle regulator
Cyclin A2 (Figure 2A). To further understand this differential
response, we conducted chromatin immunoprecipitation
(ChIP) assays. Notably, doxorubicin treatment induced pRB
recruitment to both the cell-cycle and proapoptotic promoters
(Figure 2B). Quantitative analysis of these results (see
Figure S1 available online) showed that the increase of pRB
levels was slightly higher at Caspase 7 (2-fold) and p73 (2.2fold) than at Cyclin A2 (1.5-fold) gene promoters. For all of the
other proteins that we assayed, doxorubicin treatment caused
differential changes at cell-cycle versus proapoptotic promoters
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
HDAC1 to the proapoptotic promoters in either the damaged
or undamaged cells (Figure 2B).
The coordinated enrichment of pRB, E2F1, and RNA polymerase II at the Caspase 7 and p73 promoters fits with the
hypothesis that pRB contributes to activation of proapoptotic
genes. However, we could not rule out the possibility that there
are two distinct populations of cells in which these promoters
are either bound by pRB and repressed or associated with
RNA polymerase II and activated. To address this possibility,
we performed ChIP-reChIP experiments in which immunoprecipitated pRB-chromatin complexes were eluted and then subjected to a second round of immunoprecipitation with either
control IgG or an antibody against acetyl-H4 (AcH4), a marker
of transcriptional activation (Figure 2C). As with our previous
experiment, the primary ChIP showed that doxorubicin
promoted recruitment of pRB to both the Caspase 7 and Cyclin
A2 promoters (Figure 2C). However, when we analyzed the
eluate from the pRB immunoprecipitates, we found that acetylH4 was specifically detected at the Caspase 7, but not the Cyclin
A2, gene promoter (Figure 2C). This analysis showed unequivocally that pRB was bound to the transcriptionally active Caspase
7 gene promoter, presumably via its participation in the pRBE2F1-P/CAF complex that is promoted by DNA damage. At the
same time, pRB binds to cell-cycle promoters and recruits
HDAC1 to mediate their repression.
Figure 2. DNA Damage Induces
of Proapoptotic Gene Promoters
(A) Untreated and doxo-treated asynchronously growing T98G cells were
assessed for levels of Caspase 7, p73, and Cyclin A2 mRNAs by real-time
RT-PCR analysis. Results are normalized to GAPDH and shown relative to
the levels observed in untreated cells (set to 1). Bars represent the mean of
three independent experiments ± SD.
(B) T98G cells were untreated ( ) or treated with 2 mM doxo for 16 hr (+), and
the binding of pRB, E2F1, RNA polymerase II (Pol II), and HDAC1 to the
Caspase 7, p73, and Cyclin A2 gene promoters was determined by ChIP.
(C) Acetyl-H4 (AcH4) reChIP analysis of the pRB ChIP shows that pRB is
bound to the transcriptionally active Caspase 7 gene promoter following
doxo treatment.
(Figure 2B). At the Cyclin A2 gene promoter, we found a reduction
in the binding of both E2F1 and RNA polymerase II. In addition,
the transcriptional corepressor HDAC1 was specifically recruited to the Cyclin A2 promoter in treated, but not untreated,
cells (Figure 2B). These changes are consistent with the
observed downregulation of Cyclin A2 mRNA and the prevailing
view that pRB mediates the transcriptional repression of cellcycle promoters. At the same time, doxorubicin treatment
induced the recruitment of both E2F1 (1.4- and 2.7-fold) and
RNA polymerase II (2.3- and 2-fold) to the Caspase 7 and p73
promoters. Importantly, we did not detect any recruitment of
pRB Is Required for Maximal Induction of the Apoptotic
Response In Vitro and In Vivo
These data show that pRB is associated with proapoptotic
promoters that are transcriptionally active in DNA-damaged cells.
However, they do not establish whether pRB contributes to transcriptional activation of these proapoptotic genes or to the
apoptotic response. To address these questions, we took advantage of the lentivirus pPRIME-GFP-shRB and its control pPRIMEGFP producing respectively either a short hairpin against human
pRB (shRB) or a hairpin targeting the luciferase gene, in the
context of miR30 (Stegmeier et al., 2005; http://elledgelab. We
used these viruses to infect T98G cells and selected parallel populations of GFP-positive cells. The shRB reduced pRB levels to
less than 50% of those seen in the control cells (Figure 3A).
Importantly, this partial knockdown had no effect on cell-cycle
phasing (Figure 3A). This allowed us to assess pRB’s contribution to apoptosis independent of its role in the cell cycle. We
found that this partial pRB knockdown significantly reduced
the fraction of cells undergoing apoptosis in response to either
doxorubicin or another topoisomerase II inhibitor, etoposide
(50% and 30% reduction, respectively; Figure 3C). This correlated with a reduction in the levels of p73 (>50%) and Caspase
7 (20%) mRNA in the shRB-expressing cells (Figure 3D).
Thus, we conclude that pRB loss can impair the apoptotic
response in the absence of any cell-cycle defects.
All of the previous experiments were conducted in the p53deficient tumor cell line T98G. To determine whether our findings
were more broadly relevant, we repeated this analysis in
a second tumor cell line, U2OS (Figure S2). These cells express
wild-type p53, and pRB is constitutively hyperphosphorylated
due to hypermethylation and silencing of the p16INK4a gene
promoter (Park et al., 2002). In accordance with our prior results,
Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc. 187
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
Figure 3. pRB Loss Impairs the Apoptotic
Response to DNA Damage
(A–D) T98G cells were infected with pPRIME-GFP
(GFP) or pPRIME-GFP-shRB (GFPshRB) lentivirus
and sorted for >20% GFP-positive cells.
(A–C) Sorted GFP and GFPshRB cells were
screened for the level of pRB by WB using actin
as a loading control (A), cell-cycle phasing by
FACS analysis (B), or the percentage of early
apoptotic cells by FACS (annexin V+, 7AAD ) after
culturing for 48 hr in either the absence ( ) or presence of 2 mM doxo or 25 mM etoposide (et) (C).
(D) Caspase 7 and p73 mRNA levels measured by
real-time RT-PCR analysis. Results are normalized to GAPDH and expressed relative to levels
observed in GFP-infected cells (set as 1).
(E) Wild-type (WT) or Rb2lox/2lox (cRb) mesenchymal
stem cells (MSCs) were infected with GFP- or GFPCre-expressing adenoviruses. The percentage of
GFP+ apoptotic cells was measured by FACS
(annexin V+, 7AAD ).
Bars in (B)–(E) represent the mean of three independent experiments ± SD.
we found that numerous genotoxic agents (including doxorubicin, etoposide, and camptothecin) triggered pRB to bind to
E2F1 and promoted both activation of proapoptotic and repression of cell-cycle-related E2F target genes (Figures S2A–S2C).
Moreover, pRB knockdown using either the pPRIME-GFPshRB lentivirus or a tTA-inducible RB hairpin impaired the
apoptotic response and the transcriptional activation of the
proapoptotic gene p73 (Figures S2D–S2G). Thus, pRB can
play a positive role in DNA damage-induced apoptosis in both
the absence and the presence of p53.
It has previously been reported that Rb inactivation renders
mouse embryonic fibroblasts (MEFs) more, not less, sensitive
to DNA damage-induced apoptosis (Almasan et al., 1995; Knudsen et al., 2000). We have repeated these experiments in both
germline and conditional Rb mutant MEFs and obtained similar
results (A.I. and J.A.L., unpublished data). Thus, in different
settings, pRB can either promote or inhibit apoptosis. It seemed
possible that the differential consequences of pRB loss reflect
fundamental differences between tumor versus normal, or
mouse versus human, cells. To address these possibilities, we
examined a second source of primary murine cells, mesenchymal stem cells (MSCs). We generated these MSCs from
mice carrying either wild-type (WT) or conditional Rb (cRb)
alleles, infected them with adenoviruses expressing either the
Cre recombinase gene (+Cre) or a GFP control ( Cre), and
confirmed recombination of the cRb alleles by PCR (data not
shown). The four cell populations were then treated with ionizing
radiation (Figure 3E) or doxorubicin (data not shown), and the
fraction of apoptotic cells was quantified by FACS analysis.
Cre expression had no significant effect on the level of apoptosis
in the WT cells, but it reduced apoptosis in the cRb cells by more
than 30% (Figure 3E). Thus, pRB loss also impairs the apoptotic
response of these primary murine cells.
To further extend this analysis, we next examined pRB’s role in
the DNA damage response in vivo. For this, we used mice
carrying conditional Rb alleles and a Villin-Cre transgene, which
188 Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc.
is expressed in the adult intestinal epithelium. To eliminate any
potential contribution of Cre-mediated deletion to the DNA
damage response, we selected Rb+/2lox;Villin-Cre+ mice (effectively Rb heterozygous) as controls for our analysis of Rb2lox/2lox;
Villin-Cre+ (Rb mutant) mice. Rb2lox/2lox;Villin-Cre+ mice are
known to have histologically normal intestinal crypts, but these
contain proliferating cells at ectopic locations (Kucherlapati
et al., 2006). Immunohistochemical staining confirmed that
pRB was expressed in the intestinal epithelium of the Rb+/2lox;
Villin-Cre+ controls, but not in the Rb2lox/2lox;Villin-Cre+ animals
(Figure 4A, left). We then assessed the level of proliferating cells
by screening for the cell-cycle marker Ki-67 (Figure 4A, middle).
Consistent with prior studies (Haigis et al., 2006; Kucherlapati
et al., 2006), the proliferating cells were restricted to the intestinal
crypts of the Rb+/2lox;Villin-Cre+ controls, but they existed in both
the crypts and throughout the height of the villi of Rb-deficient
intestinal epithelium. We then subjected these Rb2lox/2lox;VillinCre+ mice and their Rb+/2lox;Villin-Cre+ littermate controls to
intraperitoneal (i.p.) injections of doxorubicin and examined the
levels of apoptosis in the proximal small intestines by staining
for cleaved caspase-3 (Figure 4A, right). As expected, apoptosis
was essentially absent in the untreated intestinal epithelia of both
genotypes (Figure 4A, right). In response to treatment, we
observed high levels of cleaved caspase-3 in the Rb+/2lox;VillinCre+ control tissues. Interestingly, these apoptotic cells were
localized exclusively within the intestinal crypts, clearly correlating with the proliferative region of the intestinal epithelium.
This is consistent with the prevailing view that the cycling cells
are more predisposed to undergo apoptosis than their arrested
counterparts. Notably, quantification of 60 villi from each genotype showed that the fraction of proliferating cells undergoing
apoptosis was reduced in the Rb mutant (1.9% ± 0.88%) versus
control (3.8% ± 0.72%) tissue. This effect was most striking in
the height of the villi, where we observed few, if any, apoptotic
cells in the Rb2lox/2lox;Villin-Cre+ mice even though this zone
was highly proliferative (Figure 4A). However, we did not observe
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
Figure 4. Conditional pRB Knockout Mice Are Less Sensitive to Genotoxic Stress
(A) Analysis of proximal small intestines of Rb+/2lox;Villin-Cre+ (HET) and Rb2lox/2lox;Villin-Cre+ (MUT) mice treated with vehicle control or doxo. Left: immunostaining confirms Cre-mediated loss of pRb in the proximal small intestine of MUT animals. Middle panel: analysis of Ki-67, a marker of proliferating cells, shows that
pRB loss causes proliferation throughout the villi. Ki-67 levels were similar in the absence and presence of doxorubicin treatment. Right: staining for cleaved
caspase-3 shows high levels of apoptotic cells specifically in the base of the crypts in doxo-treated, but not untreated, WT and MUT mice. Scale bars =
50 mm in all panels.
(B) The average number of apoptotic cells per intestinal crypt (±SEM) in the proximal small intestines of doxo-treated Rb+/+;Villin-Cre+ (WT; n = 5), Rb+/2lox;VillinCre+ (HET; n = 5), and Rb2lox/2lox;Villin-Cre+ (MUT; n = 6) mice was determined by counting cleaved caspase-3-positive cells in 21 crypts for each animal.
a significant difference in the level of apoptotic cells in the
intestinal crypts of Rb2lox/2lox;Villin-Cre+ mice versus Rb+/2lox;
Villin-Cre+ controls (data not shown).
Since our cell studies had shown that a partial knockdown of
pRB was sufficient to impair the apoptotic response, we
wondered whether there might be a heterozygous mutant
phenotype in the Rb+/2lox;Villin-Cre+ intestinal crypts. To address
this possibility, we subjected a second cohort of Rb+/+;VillinCre+ (n = 5), Rb+/2lox;Villin-Cre+ (n = 5), and Rb2lox/2lox;VillinCre+ (n = 6) mice to i.p. injections of doxorubicin, stained for
cleaved caspase-3, and quantified the level of apoptosis in the
intestinal crypts. Consistent with our prior analysis, doxorubicin
treatment induced a similar apoptotic response in the intestinal
crypts of Rb+/2lox;Villin-Cre+ (HET) and Rb2lox/2lox;Villin-Cre+
(MUT) mice (Figure 4B). However, these two genotypes had
a 2-fold lower level of apoptosis than the Rb+/+;Villin-Cre+ (WT)
controls (Figure 4B). In both cases, this difference was statistically significant (p < 0.001). Thus, in this tissue, pRB loss
promotes inappropriate proliferation while reducing the ability
of these cells to undergo DNA damage-induced apoptosis.
Moreover, mutation of a single Rb allele is sufficient to impair
the apoptotic response without altering proliferation. These
observations show that Rb influences the apoptotic response
to DNA damage in vivo. They also raise the possibility that this
impaired response could occur in patients carrying germline
RB1 mutations.
E1A Promotes the Formation of Transcriptionally Active
pRB-E2F1 Complexes
Given our findings, we decided to extend our analysis to examine
the mechanism of action of adenoviral E1A. E1A is a potent
oncogene that induces uncontrolled proliferation and also sensitizes cells to apoptosis. It binds to pRB with high affinity, and this
requires the LXCXE motif that is essential for E1A’s transforming
activity (Helt and Galloway, 2003). The prevailing view is that E1A
acts to sequester pRB, allowing release of transcriptionally
active E2Fs. This model can explain the increased proliferation
rate seen for E1A-infected cells. However, prior studies have
shown that the interaction between E1A and pRB is insufficient
to promote apoptosis in response to doxorubicin (Samuelson
et al., 2005). Indeed, the previous mutant analysis suggests
that this requires E1A binding to both pRB and p400, a component of the TRAAP/Tip60 HAT chromatin-remodeling complex.
Given our observations, we hypothesized that E1A’s proapoptotic function might reflect its ability to promote formation of
transcriptionally active pRB-E2F1 complexes, in a manner analogous to genotoxic stress. To test this notion, we investigated
the interplay between E1A and the pRB-E2F1 complex using
primary IMR-90 human diploid fibroblasts that express the
murine ecotropic receptor. We selected these cells because
they apoptose in response to genotoxic agents or E1A but are
more resistant to apoptosis than many other cell lines and thus
are better able to tolerate E1A expression. Importantly, in
Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc. 189
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pRB Induces Apoptotic Genes in Response to Stress
Figure 5. pRB Facilitates E1A’s Ability to
Promote Apoptosis
(A) In the absence of E1A, a pRB-E2F1 complex
was induced by treatment of IMR-90 cells with
doxo as judged by IP for pRB and WB for pRB
and E2F1.
(B–I) IMR-90 cells were retrovirally transduced
with either pBabe-hygro vector ( E1A) or
pBabe-hygro-E1A 12S (+E1A), selected with
75 mg/ml hygromycin for 4 days, and then assayed
as follows.
(B) Cells were treated with 2 mM doxo for 16 hr, and
WB was used to determine the levels of pRB,
E2F1, E2F3A, E2F4, and E1A in pRB IPs (left)
and total lysates (right). A longer exposure of the
key input lanes is shown at far right. E1A expression strongly increased both the total levels of
pRB, E2F1, and E2F3A and the level of pRBE2F1/E2F3A complexes. At the exposure shown,
the pRB-E2F1 interaction is not visible in the
E1A +doxo cells.
(C) The binding of E1A to pRB and E2F1 is also
detected via IP of E1A.
(D) IPs with antibodies against ppRB and WB for
pRB and E2F1.
(E) The levels of pRB, E1A, E2F1, and AcH4 associated with the p73 and Cyclin A2 gene promoters
were determined by ChIP. Densitometric quantification of each signal relative to the input is shown.
(F) Real-time RT-PCR analysis of p73 and Cyclin
A2 mRNA levels. Results are expressed as arbitrary units normalized to GAPDH and show the
mean of three independent experiments ± SD.
(G–I) Control or E1A-transduced cells were infected with either GFP or GFPshRB lentiviruses.
(G) WB confirmed a reduction in pRB levels after
shRB infection, using actin as a loading control.
(H) Caspase 7, p73, and Cyclin A2 mRNA levels were determined by real-time RT-PCR analysis in cells cultured in the absence ( ) or presence of 2 mM doxo for
12 hr (+). Results are normalized to GAPDH and show the mean of three independent experiments ± SD, expressed relative to levels observed in the untreated
cells (set as 1).
(I) FACS analysis of early apoptotic cells (annexin V+, 7AAD ) after culture in the absence ( ) or presence of 1 mM etoposide (et) for 16 hr. Values represent the
percentage of apoptotic GFP+ cells and represent the mean of three independent experiments ± SD.
a manner similar to T98G and U2OS cells, doxorubicin promotes
formation of pRB-E2F1 complexes in IMR-90 cells in the
absence of E1A (Figure 5A). To study the role of E1A, IMR-90
cells were retrovirally transduced with either pBabe-puro vector
or pBabe-puro expressing the 12S form of E1A. We found that
E1A and E2F1 both coimmunoprecipitated with pRB
(Figure 5B). Doxorubicin treatment caused a modest additional
increase (1.6-fold) in the level of E2F1 coimmunoprecipitating
with pRB in the E1A-expressing IMR-90 cells (Figure 5B). These
observations show that E1A potently induces formation of pRBE2F1-E1A complexes and that exogenous DNA damage
reinforces this response.
In the presence of E1A, pRB also associated with E2F3A,
another activating E2F that is known to participate in the oncogenic stress response, but not with the repressive E2F E2F4
(Figure 5B). Importantly, the reciprocal immunoprecipitation
using antibodies against E1A also recovered both pRB and
E2F1 (Figure 5C). Since it is well established that a pocket
protein, such as pRB, is required to bridge the interaction
between E1A and E2F (Fattaey et al., 1993), we can infer that
these three proteins must be part of the same complex. Similar
190 Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc.
to our analysis of the DNA damage response, E1A also enhanced
the formation of ppRB species and their binding to E2F1
(Figure 5D). Additionally, E1A yielded a dramatic increase in
the intracellular levels of pRB, E2F1, and E2F3A, but not E2F4
(Figure 5B). DNA binding is known to protect E2F-pocket protein
complexes from degradation (Hofmann et al., 1996). Given the
observed stabilization of both pRB and the activating E2Fs in
E1A-expressing cells, we hypothesized that E1A induces the
formation of pRB-E2F1-E1A complexes that bind to DNA and
activate transcription. Thus, we used ChIP assays to assess
binding to the p73 and Cyclin A2 promoters (Figure 5E). In the
absence of E1A, we observed a significant pRB ChIP signal at
both promoters, but little or no binding of either E2F1 or acetylH4 (Figure 5E). Since the uninfected IMR-90 cells are predominantly in G0/G1 phase, we speculate that pRB contributes to
the repression of both p73 and Cyclin A2 in this setting. Accordingly, these genes appeared to be transcriptionally silent, as
judged by the lack of p73 and Cyclin A2 mRNA (Figure 5F). In
contrast, in the E1A-infected cells, pRB, E2F1, E1A, and
acetyl-H4 all associated with both the Cyclin A2 and p73
promoters, coincident with the dramatic induction of both of
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
Figure 6. Model of pRB-E2F1 Complexes
Involved in the Regulation of Proliferation
and Proapoptotic Genes in Response to
DNA Damage and E1A-Induced Oncogenic
See text for details.
these mRNAs (Figures 5E and 5F). Given the existence of a pRBE2F1-E1A complex and the positive ChIP signals for E1A, pRB,
and E2F1, we speculate that E1A’s ability to activate apoptosis
and cell-cycle genes reflects, at least in part, its direct action
at their promoters.
As noted above, the current view of E1A action is that it acts to
sequester pRB and thereby release transcriptionally active E2F.
If this model fully explains the relationship between E1A and
pRB, E1A’s ability to induce apoptosis should not be impaired
by pRB loss. To test this, we performed a pRB knockdown in
E1A-infected IMR-90 cells. The shRB yielded significant, but
not complete, pRB knockdown in both uninfected and E1Ainfected IMR-90 cells (Figure 5G). Strikingly, the shRB caused
a 2-fold reduction in the levels of Caspase 7 and p73 mRNAs
in either the absence or presence of DNA damage (Figure 5H).
Consistent with previous studies (Samuelson et al., 2005),
expression of E1A alone induced only low levels of apoptosis
in these cells, and this was unaffected by pRB levels
(Figure 5I). However, when combined with genotoxic stress,
E1A induced programmed cell death at 2-fold higher levels in
control versus shRB-expressing cells (Figure 5I). Thus, pRB
plays a positive role in E1A-induced apoptosis. Taken together,
our findings suggest that E1A associates with both pRB and
E2F1, stabilizing these proteins, and the resulting complex associates with both proapoptotic promoters to promote their transcription.
The E2F transcription factors play a key role in promoting cellular
proliferation under the control of the pRB tumor suppressor. It is
well established that E2F1, among other E2F family members,
also contributes to the induction of apoptosis in response to
either DNA damage or oncogenic stress (Iaquinta and Lees,
2007). However, the role of pRB in this process is poorly understood. We anticipated that DNA damage would have to release
E2F1 from pRB to allow it to activate proapoptotic genes.
Instead, our data suggest an unexpected mechanism of pRB
action—that DNA damage induces pRB to participate in
a transcriptionally active complex that drives expression of
proapoptotic genes. This model is supported by three central
observations: (1) pRB is induced to bind both E2F1 and the
histone acetylase P/CAF in DNA-damaged cells; (2) ChIP-reChIP
assays show unequivocally that pRB is bound to the promoters
of proapoptotic genes that are transcriptionally active; and (3)
knockdown and genetic ablation experiments confirm that pRB loss typically
reduces the apoptotic response to DNA
damage by 34%–50%. Since the
apoptotic threshold is determined by
many different factors, the degree of impairment is striking.
Moreover, this is observed in many different settings including
both primary and tumor cells derived from either mice or humans, and it is independent of p53. Thus, our finding that pRB
has proapoptotic activity has broad relevance.
The concept that pRB can participate in transcriptionally
active complexes is not without precedent, since pRB has
been shown to cooperate with differentiation-specific transcription factors in the activation of key target genes (Charles et al.,
2001; Gery et al., 2004; Thomas et al., 2001). However, this proapoptotic function of pRB is not observed in all situations. As we
outlined in the introduction, the analysis of Rb mutant embryos
led to the prevailing view that pRB is antiapoptotic. Although
much of this apoptosis is non-cell autonomous (de Bruin et al.,
2003; Wenzel et al., 2007; Wu et al., 2003), pRB loss does
promote apoptosis in a cell-autonomous manner in some tissues
of the developing embryo, and this seems to reflect an inability to
undergo terminal differentiation (de Bruin et al., 2003; Haigis
et al., 2006; Huh et al., 2004; MacPherson et al., 2003; MasonRichie et al., 2008; Ruiz et al., 2004; Wang et al., 2007; Wikenheiser-Brokamp, 2004). Moreover, some cell types, such as
MEFs, have a heightened sensitivity to DNA damage in the
absence of pRB. Thus, taken together, the existing literature
and the present study indicate that pRB can either suppress or
promote apoptosis, depending on the cellular context. We
note that there is a strong correlation between the proliferative
properties of the cell and the observed role of pRB in apoptosis.
Thus, we propose the following model of pRB action (Figure 6). In
G0/G1 cells, genotoxic stress induces pRB recruitment into the
classic repressive pRB-E2F-HDAC complex. This prevents
cell-cycle entry and thus acts indirectly to protect cells from
apoptosis. Thus, in G0/G1 cells, pRB loss would impair arrest
and thereby promote apoptosis. In contrast, in proliferating cells,
genotoxic stress favors formation of the transcriptionally active
pRB-E2F1-P/CAF complexes because the hyperphosphorylation of pRB inhibits its participation in the repressive complexes.
Consequently, in this setting, pRB is proapoptotic. Interestingly,
this context-dependent model of pRB function has the potential
to explain the well-established phenomenon that proliferating
cells have a greater predisposition to undergo apoptosis
compared to their quiescent counterparts. Importantly, both
our cell line and in vivo studies show that a reduction in pRB
levels is sufficient to impair pRB’s proapoptotic function without
any disruption of proliferation control. This was particularly
striking in our animal experiments, where Rb haploinsufficiency
Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc. 191
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
reduced the apoptotic response to doxorubicin as efficiently as
the complete inactivation of Rb. This dose-dependent effect
raises the possibility that mutation of a single Rb allele might
increase the probability of cellular transformation by impairing
the apoptotic response to DNA damage and thereby enabling
the acquisition of mutations within other genes. If this is true,
individuals carrying germline RB1 mutations would be particularly at risk.
Clearly, additional questions remain about how genotoxic
stress triggers formation of the pRB-E2F1-P/CAF complex and
directs it specifically to proapoptotic promoters. Our data
show that ppRB can participate in the DNA damage-induced
pRB-E2F1 complexes. However, it is still unclear whether pRB
phosphorylation actively promotes or is merely permissive for
formation of the proapoptotic pRB-E2F1-P/CAF complex. Moreover, although we conducted these studies using antibodies
against known Cdk phosphorylation sites (Ser780, Ser795, and
Ser807–811), we cannot be sure whether cyclin/Cdk complexes
or other kinases are responsible for this modification in the DNAdamaged cells. Furthermore, it is entirely possible that additional
posttranslational modifications of pRB and/or E2F1 may facilitate formation of the proapoptotic complex. We note that pRB
has been shown to contain a second E2F binding site that
does not interfere with E2F1’s transactivation domain (Dick
and Dyson, 2003). Thus, it is intriguing to speculate that DNA
damage somehow induces pRB and E2F1 to adopt this alternate
structure. If this model is true, this conformation must also
enable recruitment of P/CAF, which is known to be critical for
E2F1-dependent activation of proapoptotic target genes in
response to DNA damage (Ianari et al., 2004; Pediconi et al.,
Our data also cause us to revise our view of E1A’s mechanism
of action. The prevailing view holds that E1A acts to disrupt pRBE2F complexes, releasing free E2F1 to induce transcription of its
target genes. However, our data show that E1A forms a stable
complex with both pRB and the activating E2Fs. Moreover,
pRB, E2F1, and E1A are all recruited to the promoters of both
apoptosis and cell-cycle genes, coincident with their transcriptional activation. Interestingly, mutant analysis has shown that
E1A must interact with both pRB and p400 in order to promote
apoptosis in response to doxorubicin (Samuelson et al., 2005).
Thus, we now propose that E1A’s ability to promote both
apoptosis and proliferation reflects at least in part its direct
action at proapoptotic and cell-cycle gene promoters through
its association with the pRB-E2F1 complex and the concomitant
recruitment of p400 and other transcriptional coactivators
(Figure 6). Given the recent finding that oncogenic stress activates the DNA damage response (Bartkova et al., 2006; Di Micco
et al., 2006), formation of the transcriptionally active pRB-E2F1
complexes may be further reinforced through E1A’s activation
of the DNA damage-dependent process. Essentially, E1A would
trigger the DNA damage response and then piggyback onto the
resultant pRB-E2F1 complex to create a stable superactivator.
Notably, in contrast to the DNA damage-induced pRB-E2F1
complex, which specifically activates only proapoptotic genes,
the E1A-containing species has an expanded target specificity
that now includes both apoptosis and cell-cycle targets. Importantly, in agreement with this superactivator model, pRB knockdown impairs E1A’s ability to promote the transcription of both
192 Cancer Cell 15, 184–194, March 3, 2009 ª2009 Elsevier Inc.
apoptosis and cell-cycle-related genes and to induce apoptosis
in response to DNA-damaging agents.
The elucidation of the mechanism by which pRB acts as
a tumor suppressor has been complicated by various factors.
In addition to its role in cell-cycle control, pRB has been implicated in regulating a wide variety of cellular processes, including
DNA replication, differentiation, and apoptosis (Classon and
Harlow, 2002). Whereas the decreased differentiation potential
and the increase in proliferative rate observed in pRB-deficient
cells could contribute to tumorigenesis, it is more difficult to
reconcile pRB’s role as a tumor suppressor with the notion
that loss of pRB may lead to increased apoptosis. Our finding
that pRB plays a positive role in DNA damage-induced apoptosis
widens our understanding of pRB’s functions. Notably, the
behavior of pRB in the DNA damage response bears strong
parallels to that of p53: both of these tumor suppressors appear
capable of triggering either cell arrest or apoptosis, depending
on the cellular context. Given this model, we propose that RB1
inactivation in tumor cells promotes tumorigenicity by yielding
both a proliferative advantage and resistance to apoptotic stimuli
such as chemotherapeutic treatments.
Cell Culture and Infections
T98G, U2OS, and IMR-90 cell lines were cultured in DMEM with 10% heat-inactivated FBS. The IMR-90 cells overexpressed the murine ecotropic
receptor. Lentiviral and retroviral preparations and infections were performed
as described previously (Samuelson et al., 2005; Stegmeier et al., 2005).
Mesenchymal stem cells were generated by mechanically crushing femurs
and tibias of 6- to 8-week-old mice and culturing in a-MEM with 10% heatinactivated FBS. These cells were infected with either Ad5CMVCre-eGFP or
Ad5CMVeGFP at about 100 plaque-forming units per cell for 4 hr (University
of Iowa Gene Transfer Vector Core) and treated 3 days later with 2 mM doxorubicin or irradiated for 15 min for a total dose of 10 Gy and analyzed by FACS
after 24 hr. Hairpins used in this study are shown in Table S1.
FACS Analysis
Suspensions of T98G or IMR90 cells were processed for DNA content as
described previously (Pozarowski and Darzynkiewicz, 2004). For apoptosis
assays, cell suspensions were stained with annexin V APC and 7AAD (Becton
Dickinson). Cells were analyzed using a FACScan system (Becton Dickinson),
and the data were analyzed using ModFit LT software (Verity Software).
Immunoprecipitations and Western Blotting
Proteins were extracted with RIPA buffer (Pediconi et al., 2003) and quantified
using BCA protein assay reagent (Pierce). Extracts were immunoprecipitated
with the indicated antibodies and either protein A or protein G Plus (Santa Cruz
Biotechnology). Antibodies were obtained from Santa Cruz Biotechnology
(E2F1 [C-20], RNAPol-II [N-20], E1A [M73-HRP], actin [I-19], E2F3 [C-18],
and E2F4 [C-20]), Cell Signaling Technology (RB [4-H1], 780-795-807-811
ppRB, and cleaved caspase-3), BD Pharmingen (pRB and Ki-67), Upstate
Biotechnology (acetyl-H4 and HDAC1), and P. Nakatani of Dana-Farber
Cancer Institute, Harvard Medical School (P/CAF) .
ChIP Assay
Chromatin immunoprecipitation was performed as described previously
(Pediconi et al., 2003). For reChIP experiments, RB immunoprecipitates
were eluted with DTT and then subjected to a second round of immunoprecipitation with acetyl-H4 antibody or with IgG. Densitometric quantification of
ChIP results was performed using the NIH ImageJ 1.4 program. Primer
sequences are described in Table S2.
Cancer Cell
pRB Induces Apoptotic Genes in Response to Stress
Real-Time RT PCR
Total RNA was extracted with a QIAGEN RNeasy Kit and reverse transcribed
with oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen).
Quantitative analysis of Caspase 7, TAp73, and Cyclin A2 mRNA expression
was performed employing an ABI PRISM 7900 Sequence Detection System
(Applied Biosystems). Gene expression values were normalized to GAPDH.
Results are expressed as mean ± SD. Statistical differences were analyzed
by Mann-Whitney nonparametric test. p < 0.05 was considered statistically
Animal Maintenance and Tissue Analyses
The Rb2lox;Villin-Cre+ mice were maintained and genotyped as described
previously (Haigis et al., 2006). The relevant genotypes were injected intraperitoneally with either saline vehicle (0.9% NaCl) or doxorubicin (10 mg/kg) and
sacrificed 3 hr later, and intestines were collected for histology. All animal
procedures followed protocols approved by MIT’s Committee on Animal
Care. pRB immunostaining was conducted using an UltraVision LP Detection
System (Lab Vision Corporation) with the primary antibody (G3-245, BD) at
a concentration of 1:100. Immunohistochemistry was performed as described
previously for cleaved caspase-3 (Haigis et al., 2006) and Ki-67 (Danielian
et al., 2007). All samples were counterstained with hematoxylin.
The Supplemental Data include two tables and two figures and can be
found with this article online at
Vectors and cell lines were kindly provided by F. Stegmeier (pPRIME-CMVGFP and pPRIME-CMV-GFP-shRB) and S. Lowe (pBabe-hygro, pBabeE1A-hygro, and U2OS rtTA RB.670). We thank K. Hilgendorf and A. Cheung
for technical help and M. Levrero, Koch Institute colleagues, and members
of the Gulino and Lees laboratories for helpful discussions throughout this
work. This project was supported by grants from the Associazione Italiana
per la Ricerca sul Cancro; Telethon (GGP07118); Istituto Pasteur Cenci Bolognetti; Ministero dell’Istruzione, Università€ e della Ricerca; the Italian Ministry of
Health; and the Rome Oncogenomic Center to A.G. and from the NIH (2-P01CA42063) to J.A.L. A.I. was supported by postdoctoral fellowships from the
American-Italian Cancer Foundation and from the Marie Curie Outgoing International Fellowship. J.A.L. is a Ludwig Scholar at MIT.
Received: March 20, 2008
Revised: August 3, 2008
Accepted: January 26, 2009
Published: March 2, 2009
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